The ratio of intracellular CRY proteins determines the clock period length

Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics of the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period. •Rhythmic CRY2, like CRY1, in the correct CRY1 phase is sufficient to rescue clock oscillation in CRY null cells.•The short-period mammalian CRY2 protein is more stable than the CRY1 protein.•The N-terminal polypeptide of CRY2 contributes to its stability and Per2 repression, but it does not affect the period.•The C-terminal tails of CRYs regulate their protein stability and nuclear import, but the import rate governs the period.•The ratio, rather than the absolute amounts of CRY1 and CRY2 proteins, determines the period in mammalian cells.